Abstract

Background: Since fibroblasts are the predominant stromal cell type in colorectal carcinomas (CRC), it is reasonable to assume that fibroblast-tumor cell crosstalk play an important role in the production and maintenance of the malignant phenotype. Thus, it is very relevant to abort the supply of such factors responsible for this crosstalk in order to hamper tumor growth.

In this work, we studied the crosstalk between Normal Colonic Fibroblasts (NCF) and tumor cells, its relation with de novo chemoresistance and fibroblast activation, providing hints for the treatment of CRC.

Methods: After 5 days of Transwell coculture between fibroblasts and tumor cells we obtained mRNA of each cell type to obtain differentially expressed genes compared to monocultures (Affymetrix GeneChip Human Gene 1.0 ST Array). From genes with fold change>2, we selected those codifying for secreted or membrane proteins in both tumor cells and fibroblast. Protein-protein interaction (PPI) analysis was carried out by means of BIANA software searching for fibroblast secreted proteins with receptors in tumor cells plasma membrane and viceversa. We also studied the main pathways involved in such crosstalk in control conditions or pharmacologically disrupted. In addition we performed functional assays (proliferation, migration and citotoxicity against Oxaliplatin and 5FU). We also performed a cytokine array (Raybiotech) to characterise products mediating such crosstalk.

Results: After integrating our transcriptomic/PPI data with information obtained by means of GSEA we stated our working hypothesis as: cytokines released by NCF stimulate tumor cells to secrete IL1β and TGFβ1. These two soluble factors are responsible for the NCF activation. Activated fibroblasts in turn began to produce large amounts of IL1β/TGFβ1 targets, mainly chemokines, cytokines, EGFR-ligands and PAI-1, signalling basically through IL6R, CCR2 (JAK-STAT pathway), CCR6, EGFR and CXCR3 (PI3K-AKT pathway), pathways involved in chemoresistance in CRC. Hampering the crosstalk by means of a blocking IL1β antibody and TGFBR1 inhibitor sensitizes tumor cells to Oxaliplatin and 5FU and induces a change in the cytokines mediating the crosstalk. Cell signalling reveals an activation of non-canonical TGFβ1 evidenced by TAK1 phosphorilation and NFkβ activation.

Conclusions: Disrupting the crosstalk between fibroblasts and tumor cells with TGFBR1 inhibitor and a blocking IL1β antibody induces a change in the cytokine repertoire of both cells that activates NFkβ pathway in fibroblasts and a sustained activation of AKT in tumor cells. We can avoid such activation inhibiting non-canonical TGFβ pathway with a TAK1 inhibitor. The combined use of canonical and non-canonical TGFβ inhibitors could an interesting approach to overcome microenvironment-mediated drug resistance.